M. tuberculosis (Mtb) causes latent infections that affect a third of the world's population and active tuberculosis kills two million people every year. Chemotherapy of tuberculosis requires long treatment regimens and is complicated by the emergence of multi-drug resistant and extensively drug resistant Mtb strains. New drugs that shorten TB chemotherapy and cure drug resistant TB are urgently needed. Mtb encounters an acidic pH within phagosomes of interferon-3 activated macrophages and must prevent excessive entry of protons into its cytosol. One pathway important for pH homeostasis and virulence of Mtb depends on the membrane-associated serine hydrolase Rv3671c. The goals of this proposal are to determine the molecular mechanisms by which Rv3671c protects Mtb from intracellular acidification and contributes to virulence. Our specific aims are: I. To determine why Rv3671c is required for resistant of Mtb against acid. We will investigate the mechanism by which this serine hydrolase controls pH and acid resistance. II. To determine why Rv3671c is required for persistence in activated macrophages and mice. We will investigate if Rv3671c affects phagosome maturation in IFN-3 activated macrophages. We will use genetically altered mice to determine if Rv3671c protects against host defense mechanisms, which act synergistically with phagosome acidification. III. To identify small molecules that interfere with pH homeostasis in Mtb. We will use a high- throughput screen to identify such inhibitors, which will help to investigate the biology of intrabacterial pH homeostasis in Mtb and facilitate the development of new drugs against tuberculosis.